Hi! I'm working with MSC from Umbilical Cord for exosome isolation. I used beta amyloid to induce/increase the exosome production and normal culture conditions to compare. I'm using UC protocol: 2,000 xg/30 min; 25,000 xg/30 min; 110,000 xg/4 hrs; 110,000 xg/1.5 hrs. We obtain a visible cloudy pellet difficult to resuspend in PBS.
However, we have several issues:
- The protein content measured by DC assay kit is different each time we quantify
- In DLS, we obtain two peaks with an average size of 120 nm and 500 nm
We thought it was a matter of aggregation, but we tried to resuspend vigorously with up and down pippeting and vortexing and even resuspending in DPBS. The protein concentration did not improved.
We re-concentrate 3 samples in one with an extra UC 110,000 xg/1.5 hrs, but protein quantification decrease (instead of the opposite expected result). Surprisingly, in the DLS size measure, we obtain only one peak with an average size of 100 nm
I don't know whats happening... Did anyone has an opinion?
Dear Lilia,
I suggest that you read the MISEV Check List for exosome production and measurements to find answers to your questions. https://pubmed.ncbi.nlm.nih.gov/30637094/
It may be helpful.
Best wishes,
Hi Lilia,
DLS is very susceptible to polydispersity, and sample quality, which can both explain the difference in size distribution between the two sets of UC.
Instead of relying on protein assays, and as indicated by Gulderen Yanikkaya Demirel , I would suggest targeting specific EV markers to assess the effectiveness of your isolation protocol. WB or flow cytometry protocols are well described in the literature. Once you have confirmed that your isolate contains EVs, you can take steps to improve yield and purity, if that's your objective. At this stage, DLS might become more consistant, though single-particle analysis by NTA, RPS or nano-flow will be more relevant, as well as quantitative.
Best wishes
Dimitri
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